Skeletal muscle trauma often leads to severe functional deficits due to insufficient regeneration of the musculature. Approaches to reduce the collateral damage due to soft tissue trauma have until recently been predominantly conservative and based on preparing the muscular macro-structure to enable intrinsic healing mechanisms of the muscle. Such approaches include the traditional RICE approach (Rest Ice Compression Elevation) and the suturing of retracted muscle stumps, which allow the intrinsic regenerative capacities of the muscle to take effect. The central process of such intrinsic mechanisms is represented by the activation of satellite cells and their contribution to new myofibers (Mauro, A., J Biophys Biochem Cytol 9, 493, 1961).
A relatively recent concept in muscle and tissue regeneration is based on the idea of introducing progenitor cells or other cells capable of inducing or undergoing regeneration at the site of muscle or tissue injury. Cells with regenerative potential have been proposed to be able to stimulate the growth and healing process of human tissues. Mechanisms of this stimulation are on the one hand a secretion of trophic factors and of immunomodulatory factors and on the other hand a contribution to contractile muscle fibers by the progenitor cells.
Mesenchymal stem cell (MSC) therapy offers an effective tool in the regeneration of muscle function after severe trauma. MSCs are mesenchymal cells found, e.g. in bone marrow, dermis, placenta, adipose tissue and periosteum. A part of these cells are capable of differentiating into specific types of mesenchymal or connective tissues including adipose, osseous, cartilaginous, muscular tissue and elastic and fibrous connective tissues.
MSCs exhibit a high proliferation potential and lower donor site morbidity when compared to muscle-derived cells, thus rendering them an ideal candidate for clinical application in muscle regeneration.
New therapeutic possibilities have recently been proposed for muscle regeneration that are based on the idea of an extrinsic support of the cellular regeneration by increasing the number of progenitor cells in the injured tissue. Initial experiments showed that the transplantation of myoblasts could partially restore dystrophin in myofibers of musculature deficient of this protein (Partridge, T. A., et al. Nature 337, 176, 1989, Huard, J., et al. Clin Sci (Lond) 81, 287, 1991) and improve the condition of regenerating denervated and ischemic musculature compared to undamaged control muscles (DeRosimo, J. F., et al. Cell Transplant 9, 369, 2000, Arcila, M. E., et al. J Neurobiol 33, 185, 1997).
The clinical application of MSCs in skeletal muscle regeneration in the prior art is limited. To date, few experimental studies have investigated the effect of local MSC transfer on skeletal muscle regeneration, two of which evaluated the functional outcome of cellular therapy (Matziolis, G., et al. Tissue Eng 12, 361, 2006, Natsu, K., et al. Tissue Eng 10, 1093, 2004).
Previous studies have investigated the effect of MSCs in treating toxin-damaged muscle tissue. For example, genetically modified human mesenchymal stem cells were injected in dystrophin deficient musculature of immunosuppressed mice after toxic damage. No improvement in muscle contraction forces could be found and the authors stated that MSCs were in fact currently unsuitable for the treatment of muscle dystrophies (Gang et al. Experimental Cell Research 315: 2624, 2009). Alternative cell types have also been investigated in treating skeletal muscle injury. The transplantation of human umbilical cord blood cells into crushed gastrocnemius muscles demonstrated little change in muscle regeneration, whereby no functional effect in muscle regeneration was shown (Brzoska et al, Experimental Hematology, 2006).
The applicant has previously demonstrated that the local transplantation of 1×106 autologous MSCs one week after an injury to the soleus muscle could improve muscle forces measured three weeks after application (Matziolis, G., et al. Tissue Eng 12, 361, 2006). The functional effect of the MSCs increases with the transplanted cell number. The one week delay between injury to the soleus muscle and local injection of MSCs adhered to the commonly accepted paradigm that the initial inflammatory phase, with a high activity of macrophages, would be disadvantageous for the putative integration and regenerative effect of the applied MSCs (Jarvinen, M. and Sorvari, T. Acta Pathol Microbiol Scand [A] 83, 259, 1975, Li, Y. and Huard, J. Am J Pathol 161, 895, 2002).
The biological processes which are triggered and maintained by an injury to skeletal muscle tissue are diverse. The natural course following a skeletal muscle injury is initiated by an inflammatory phase, which is determined by the invasion of neutrophils and later macrophages (see FIG. 1, adapted from Li, Y. and Huard, J. Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle, Am J Pathol 161, 895, 2002). The presence of inflammatory cells within the tissue starts immediately after injury and lasts up to several weeks. Their initial task is to remove necrotic tissue to enable regenerative processes, which begin with the activation of satellite cells already from the third day after muscle fiber damage.
The prior art which relates to mechanisms of muscular regeneration by stem cells is particularly focused on the paracrine regeneration stimulating or immunomodulatory effect of the transplanted cells, on the impact of fusion of the cells with myofibers or a differentiation into myocytes. The first most likely improves the survival of myocytes within the target tissue and on the other hand might also decrease the collateral damage, which is effected by local inflammation.
Compared to the immediate injection, MSCs that are locally transplanted seven days after injury are confronted with some inflammation in the muscle tissue, but to a significantly lower extent. At this time regenerating myofibers are predominant, although collagenous fibrous tissue has already been deposited in defect zones and in the interstitium.
The ideal time to transplant progenitor cells for the regeneration of skeletal muscle damage has not been established. To our knowledge, previous work on skeletal muscular regeneration has provided no recommendation regarding application time point due to the lack of studies aimed at this question. Previous reports, describing the transplantation of human umbilical cord blood cells into crushed gastrocnemius muscles of immunosuppressed mice or the transplantation of myoblasts into dystrophin deficient muscles after toxin injury could not demonstrate any therapeutic effect leading to the assumption that the immediate transplantation of progenitor cells is prone to a worse outcome because of the initial hostile microenvironment (Gang et al. Experimental Cell Research 315: 2624, 2009; Rousseau al. Cell Transplant. 19(5), 589, 2010).
The general view found in the prior art is that MSCs transplanted directly after muscle trauma would not be able to exert their effect on regeneration because of the hostile microenvironment. The astonishing aspect of the present invention is that an immediate application of MSCs after a muscle injury is equivalently capable of increasing muscle contraction forces as a delayed transplantation. The present invention provides a more efficient and more amenable alternative method for the local transplantation of MSCs, characterized in that the MSCs are applied immediately or shortly after skeletal muscle injury. The prior art method of transplantation one week after injury presents multiple disadvantages regarding patient discomfort and risk. The unexpected effect that muscle regeneration is effective after immediate MSC transplantation is of enormous pragmatic importance as the transplantation of MSCs directly after muscle trauma reflects current clinical demands.